Recombinant proteins are an emerging class of therapeutic agents. Such recombinant therapeutics have engendered advances in protein formulation and chemical modification. Such modifications can protect therapeutic proteins, primarily by blocking their exposure to proteolytic enzymes. Protein modifications may also increase the therapeutic protein's stability, circulation time, and biological activity. A review article describing protein modification and fusion proteins is Francis (1992), Focus on Growth Factors 3:4–10 (Mediscript, London), which is hereby incorporated by reference.
One useful modification is combination with the “Fc” domain of an antibody. Antibodies comprise two functionally independent parts, a variable domain known as “Fab”, which binds antigen, and a constant domain known as “Fc”, which links to such effector functions as complement activation and attack by phagocytic cells. An Fc has a long serum half-life, whereas an Fab is short-lived. Capon et al. (1989), Nature 337: 525–31. When constructed together with a therapeutic protein, an Fc domain can provide longer half-life or incorporate such functions as Fc receptor binding, protein A binding, complement fixation and perhaps even placental transfer. Id. Table 1 summarizes use of Fc fusions known in the art.
TABLE 1Fc fusion with therapeutic proteinsFusionTherapeuticForm of FcpartnerimplicationsReferenceIgG1N-terminus ofHodgkin's disease;U.S. Pat. No.CD30-Lanaplastic lymphoma; T-5,480,981cell leukemiaMurine Fcγ2aIL-10anti-inflammatory;Zheng et al. (1995), J. Immunol.transplant rejection154: 5590–600IgG1TNF receptorseptic shockFisher et al. (1996), N. Engl.J. Med. 334: 1697–1702;Van Zee, K. et al.(1996), J. Immunol. 156:2221–30IgG, IgA,TNF receptorinflammation,U.S. Pat. No. 5,808,029,IgM, or IgEautoimmune disordersissued Sep. 15, 1998(excludingthe firstdomain)IgG1CD4 receptorAIDSCapon et al. (1989),Nature 337: 525–31IgG1,N-terminusanti-cancer, antiviralHarvill et al. (1995),IgG3of IL-2Immunotech. 1: 95–105IgG1C-terminus ofosteoarthritis;WO 97/23614, publishedOPGbone densityJul. 3, 1997IgG1N-terminus ofanti-obesityPCT/US 97/23183, filedleptinDec. 11, 1997Human IgCTLA-4autoimmune disordersLinsley (1991), J. Exp.Cγ1Med. 174: 561–9
A much different approach to development of therapeutic agents is peptide library screening. The interaction of a protein ligand with its receptor often takes place at a relatively large interface. However, as demonstrated for human growth hormone and its receptor, only a few key residues at the interface contribute to most of the binding energy. Clackson et al. (1995), Science 267: 383–6. The bulk of the protein ligand merely displays the binding epitopes in the right topology or serves functions unrelated to binding. Thus, molecules of only “peptide” length (2 to 40 amino acids) can bind to the receptor protein of a given large protein ligand. Such peptides may mimic the bioactivity of the large protein ligand (“peptide agonists”) or, through competitive binding, inhibit the bioactivity of the large protein ligand (“peptide antagonists”).
Phage display peptide libraries have emerged as a powerful method in identifying such peptide agonists and antagonists. See, for example, Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science 249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No. 5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar. 12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No. 5,338,665, issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul. 13, 1999; WO 96/40987, published Dec. 19, 1996; and WO 98/15833, published Apr. 16, 1998 (each of which is incorporated by reference). In such libraries, random peptide sequences are displayed by fusion with coat proteins of filamentous phage. Typically, the displayed peptides are affinity-eluted against an antibody-immobilized extracellular domain of a receptor. The retained phages may be enriched by successive rounds of affinity purification and repropagation. The best binding peptides may be sequenced to identify key residues within one or more structurally related families of peptides. See, e.g., Cwirla et al. (1997), Science 276: 1696–9, in which two distinct families were identified. The peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401–24.
Structural analysis of protein-protein interaction may also be used to suggest peptides that mimic the binding activity of large protein ligands. In such an analysis, the crystal structure may suggest the identity and relative orientation of critical residues of the large protein ligand, from which a peptide may be designed. See, e.g., Takasaki et al. (1997), Nature Biotech. 15: 1266–70. These analytical methods may also be used to investigate the interaction between a receptor protein and peptides selected by phage display, which may suggest further modification of the peptides to increase binding affinity.
Other methods compete with phage display in peptide research. A peptide library can be fused to the carboxyl terminus of the lac repressor and expressed in E. coli. Another E. coli-based method allows display on the cell's outer membrane by fusion with a peptidoglycan-associated lipoprotein (PAL). Hereinafter, these and related methods are collectively referred to as “E. coli display.” In another method, translation of random RNA is halted prior to ribosome release, resulting in a library of polypeptides with their associated RNA still attached. Hereinafter, this and related methods are collectively referred to as “ribosome display.” Other methods employ chemical linkage of peptides to RNA; see, for example, Roberts & Szostak (1997), Proc. Natl. Acad. Sci. USA, 94:12297–303. Hereinafter, this and related methods are collectively referred to as “RNA-peptide screening.” Chemically derived peptide libraries have been developed in which peptides are immobilized on stable, non-biological materials, such as polyethylene rods or solvent-permeable resins. Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass slides. Hereinafter, these and related methods are collectively referred to as “chemical-peptide screening.” Chemical-peptide screening may be advantageous in that it allows use of D-amino acids and other unnatural analogues, as well as non-peptide elements. Both biological and chemical methods are reviewed in Wells & Lowman (1992), Curr. Opin. Biotechnol. 3: 355–62.
Conceptually, one may discover peptide mimetics of any protein using phage display and the other methods mentioned above. These methods have been used for epitope mapping, for identification of critical amino acids in protein-protein interactions, and as leads for the discovery of new therapeutic agents. E.g., Cortese et al. (1996), Curr. Opin. Biotech. 7: 616–21. Peptide libraries are now being used most often in immunological studies, such as epitope mapping. Kreeger (1996), The Scientist 10(13): 19–20.
Of particular interest here is use of peptide libraries and other techniques in the discovery of pharmacologically active peptides. A number of such peptides identified in the art are summarized in Table 2. The peptides are described in the listed publications, each of which is hereby incorporated by reference. The pharmacologic activity of the peptides is described, and in many instances is followed by a shorthand term therefor in parentheses. Some of these peptides have been modified (e.g., to form C-terminally cross-linked dimers). Typically, peptide libraries were screened for binding to a receptor for a pharmacologically active protein (e.g., EPO receptor). In at least one instance (CTLA4), the peptide library was screened for binding to a monclonal antibody.
TABLE 2Pharmacologically active peptidesBindingpartner/Form ofprotein ofPharmacologicpeptideinterestaactivityReferenceintrapeptideEPO receptorEPO-mimeticWrighton et al. (1996),disulfide-Science 273: 458–63;bondedU.S. Pat. No. 5,773,569,issued Jun. 30, 1998 toWrighton et al.C-terminallyEPO receptorEPO-mimeticLivnah et al. (1996),cross-linkedScience 273: 464–71;dimerWrighton et al. (1997),Nature Biotechnology 15:1261–5; Internationalpatent application WO96/40772, publishedDec. 19, 1996linearEPO receptorEPO-mimeticNaranda et al. (1999),Proc. Natl. Acad. Sci.USA, 96: 7569–74linearc-MplTPO-mimeticCwirla et al. (1997)Science 276: 1696–9;U.S. Pat. No. 5,869,451,issued Feb. 9, 1999; U.S.Pat. No. 5,932,946,issued Aug. 3, 1999C-terminallyc-MplTPO-mimeticCwirla et al. (1997),cross-linkedScience 276: 1696–9dimerdisulfide-stimulation ofPaukovits et al. (1984),linked dimerhematopoiesisHoppe-Seylers Z.(“G-CSF-mimetic”)Physiol. Chem. 365: 303–11;Laerum et al. (1988),Exp. Hemat. 16: 274–80alkylene-G-CSF-mimeticBhatnagar et al. (1996),linked dimerJ. Med. Chem. 39: 3814–9;Cuthbertson et al.(1997), J. Med. Chem.40: 2876–82; King et al.(1991), Exp. Hematol.19: 481; King et al.(1995), Blood 86 (Suppl.1): 309alinearIL-1 receptorinflammatory andU.S Pat. No. 5,608,035;autoimmune diseasesU.S. Pat. No. 5,786,331;(“IL-1 antagonist” orU.S. Pat. No. 5,880,096;“IL-1ra-mimetic”)Yanofsky et al. (1996),Proc. Natl. Acad. Sci. 93:7381–6; Akeson et al.(1996), J. Biol. Chem.271: 30517–23;Wiekzorek et al. (1997),Pol. J. Pharmacol. 49:107–17; Yanofsky (1996),PNAs, 93: 7381–7386.linearFacteurstimulation ofInagaki-Ohara et al.thymiquelymphocytes(1996), Cellular Immunol.serique (FTS)(“FTS-mimetic”)171: 30–40; Yoshida(1984), Int. J. Immunopharmacol,6: 141–6.intrapeptideCTLA4 MAbCTLA4-mimeticFukumoto et al. (1998),disulfideNature Biotech. 16: 267–70bondedexocyclicTNF-α receptorTNF-α antagonistTakasaki et al. (1997),Nature Biotech. 15: 1266–70;WO 98/53842,published Dec. 3, 1998linearTNF-α receptorTNF-α antagonistChirinos-Rojas ( ), J. Imm.,5621–5626.intrapeptideC3binhibition of complementSahu et al. (1996), J. Immunol.disulfideactivation; autoimmune157: 884–91;bondeddiseasesMorikis et al. (1998),(“C3b-antagonist”)Protein Sci. 7: 619–27linearvinculincell adhesion processes -Adey et al. (1997),cell growth, differentiation,Biochem. J. 324: 523–8wound healing, tumormetastasis (“vinculinbinding”)linearC4 bindinganti-thromboticLinse et al. (1997), J. Biol. Chem.protein (C4BP)272: 14658–65linearurokinaseprocesses associated withGoodson et al. (1994),receptorurokinase interaction withProc. Natl. Acad. Sci. 91:its receptor (e.g.,7129–33; Internationalangiogenesis, tumor cellapplication WOinvasion and metastasis);97/35969, published(“UKR antagonist”)Oct. 2, 1997linearMdm2, Hdm2Inhibition of inactivation ofPicksley et al. (1994),p53 mediated by Mdm2 orOncogene 9: 2523–9;hdm2; anti-tumorBottger et al. (1997) J. Mol. Biol.(“Mdm/hdm antagonist”)269: 744–56;Bottger et al. (1996),Oncogene 13: 2141–7linearp21WAF1anti-tumor by mimickingBall et al. (1997), Curr.the activity of p21WAF1Biol. 7: 71–80linearfarnesylanti-cancer by preventingGibbs et al. (1994), Celltransferaseactivation of ras oncogene77: 175–178linearRas effectoranti-cancer by inhibitingMoodie et al. (1994),domainbiological function of theTrends Genet 10: 44–48ras oncogeneRodriguez et al. (1994),Nature 370: 527–532linearSH2/SH3anti-cancer by inhibitingPawson et al (1993),domainstumor growth withCurr. Biol. 3: 434–432activated tyrosine kinasesYu et al. (1994), Cell76: 933–945linearp16INK4anti-cancer by mimickingFåhraeus et al. (1996),activity of p16; e.g.,Curr. Biol. 6: 84–91inhibiting cyclin D-Cdkcomplex (“p16-mimetic”)linearSrc, Lyninhibition of Mast cellStauffer et al. (1997),activation, IgE-relatedBiochem. 36: 9388–94conditions, type Ihypersensitivity (“Mastcell antagonist”)linearMast celltreatment of inflammatoryInternational applicationproteasedisorders mediated byWO 98/33812, publishedrelease of tryptase-6Aug. 6, 1998(“Mast cell proteaseinhibitors”)linearSH3 domainstreatment of SH3-Rickles et al. (1994),mediated disease statesEMBO J. 13: 5598–5604;(“SH3 antagonist”)Sparks et al. (1994), J. Biol. Chem.269: 23853–6;Sparks et al. (1996),Proc. Natl. Acad. Sci. 93:1540–4linearHBV coretreatment of HBV viralDyson & Muray (1995),antigen (HBcAg)infections (“anti-HBV”)Proc. Natl. Acad. Sci. 92:2194–8linearselectinsneutrophil adhesion;Martens et al. (1995), J. Biol. Chem.inflammatory diseases270: 21129–36;(“selectin antagonist”)European patentapplication EP 0 714912, published Jun. 5, 1996linear,calmodulincalmodulin antagonistPierce et al. (1995),cyclizedMolec. Diversity 1: 259–65Dedman et al.(1993), J. Biol. Chem.268: 23025–30; Adey & Kay(1996), Gene 169:133–4linear,integrinstumor-homing; treatmentInternational applicationscyclized-for conditions related toWO 95/14714, publishedintegrin-mediated cellularJun. 1, 1995; WOevents, including platelet97/08203, publishedaggregation, thrombosis,Mar. 6, 1997; WOwound healing,98/10795, publishedosteoporosis, tissueMar. 19, 1998; WOrepair, angiogenesis (e.g.,99/24462, published Mayfor treatment of cancer),20, 1999; Kraft et al.and tumor invasion(1999), J. Biol. Chem.(“integrin-binding”)274: 1979–1985cyclic, linearfibronectin andtreatment of inflammatoryWO 98/09985,extracellularand autoimmunepublished Mar. 12, 1998matrixconditionscomponents ofT cells andmacrophageslinearsomatostatintreatment or prevention ofEuropean patentand cortistatinhormone-producingapplication 0 911 393,tumors, acromegaly,published Apr. 8, 1999giantism, dementia,gastric ulcer, tumorgrowth, inhibition ofhormone secretion,modulation of sleep orneural activitylinearbacterialantibiotic; septic shock;U.S. Pat. No. 5,877,151,lipopolysaccharidedisorders modulatable byissued Mar. 2, 1999CAP37linear orpardaxin,antipathogenicWO 97/31019, publishedcyclic,mellitinAug. 28, 1997including D-amino acidslinear, cyclicVIPimpotence,WO 97/40070, publishedneurodegenerativeOct. 30, 1997disorderslinearCTLscancerEP 0 770 624, publishedMay 2, 1997linearTHF-gamma2Burnstein (1988),Biochem., 27: 4066–71.linearAmylinCooper (1987), Proc.Natl. Acad. Sci.,84: 8628–32.linearAdrenomedullinKitamura (1993), BBRC,192: 553–60.cyclic, linearVEGFanti-angiogenic; cancer,Fairbrother (1998),rheumatoid arthritis,Biochem., 37: 17754–17764.diabetic retinopathy,psoriasis (“VEGFantagonist”)cyclicMMPinflammation andKoivunen (1999), Natureautoimmune disorders;Biotech., 17: 768–774.tumor growth(“MMP inhibitor”)HGH fragmentU.S. Pat. No. 5,869,452Echistatininhibition of plateletGan (1988), J. Biol. Chem.,aggregation263: 19827–32.linearSLESLEWO 96/30057, publishedautoantibodyOct. 3, 1996GD1alphasuppression of tumorIshikawa et al. (1998),metastasisFEBS Lett. 441 (1): 20–4antiphospholipidendothelial cell activation,Blank et al. (1999), Proc.beta-2-antiphospholipidNatl. Acad. Sci. USA 96:glycoprotein-isyndrome (APS),5164–8(β2GPI)thromboembolicantibodiesphenomena,thrombocytopenia, andrecurrent fetal losslinearT Cell ReceptordiabetesWO 96/11214, publishedbeta chainApr. 18, 1996aThe protein listed in this column may be bound by the associated peptide (e.g., EPO receptor, IL-1 receptor) or mimicked by the associated peptide. The references listed for each clarify whether the molecule is bound by or mimicked by the peptides.bFTS is a thymic hormone mimicked by the molecule of this invention rather than a receptor bound by the molecule of this invention.
Peptides identified by peptide library screening have been regarded as “leads” in development of therapeutic agents rather than as therapeutic agents themselves. Like other proteins and peptides, they would be rapidly removed in vivo either by renal filtration, cellular clearance mechanisms in the reticuloendothelial system, or proteolytic degradation. Francis (1992), Focus on Growth Factors 3: 4–11. As a result, the art presently uses the identified peptides to validate drug targets or as scaffolds for design of organic compounds that might not have been as easily or as quickly identified through chemical library screening. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401–24; Kay et al. (1998), Drug Disc. Today 3: 370–8. The art would benefit from a process by which such peptides could more readily yield therapeutic agents.